Project Summary/Abstract The primary goal of this project is to use cryo-EM single particle analysis to obtain atomic resolution structures (2.2-2.9 ) of the functional lethal anthrax toxin containing the N terminal domain lethal factor (LFN) bound to the anthrax toxin protective antigen (PA) pore complexes inserted into POPC lipid nanodiscs (LFN-PA pore-nanodiscs). This complex, as well as the PA-pore nanodisc complex, are exposed to translocation competent pH conditions (5.5) and the atomic structures of each of these single LFN or 3 bound LFN to the anthrax pore will be evaluated to uncover the dynamic differences between translocation competent (pH 5.0). These forms will be compared with the inhibited (pH 7.5) forms. Our preliminary structure with a single data collection session on Hong Zhou's Titan Krios yielded a 5.9 A structure that appears to show extra density from the LFN unstructured tail associated with the phe clamp pore lumen. More high resolution data is required to obtain an atomic resolution structures will be obtained from in collaboration with Hong Zhou group (UCLA) (prescreening with Tommi White- MU). We routinely obtain superior solubilized nanodisc (lipid bilayer) complexes in vitreous ice of both 1LFN-PA pore and saturated 3LFN-PA pore complexes to at pH 5.5 enabling us to observe the pH induced changes in atomic structures of these PA pore complexes. Our previously NIH R01 funded structural analysis with an CCD detector (Wah Chiu NCMI site Baylor) culminated in a complete 17 3:1 LFN-PA pore nanodisc complex structure. Acquisition of the desired molecular detail of these initial engagement complexes will specifically provide the atomic snapshots of complexes in various physiologically relevant pH environments. Research and Biotechnology implications: ? Our novel methods to generate cryo-EM ready samples and acquire atomic structure of the PA pore translocon nanodisc complexes should serve as a reasonable template to enhance the acquisition of atomic structure for other aggregation prone membrane proteins inserted into authentic lipid bilayers. ? Our approaches allow us to both control initial ligand association complexes for easier sample preparation of 1:1 and 3:1 (LFN:PA pore) complexes and specific population selection methodologies for cryo-EM analysis. Our research represents the first successes in constructing late endosomal anthrax complexes at pH 5.0 using novel solution protocols. Our approach is highly relevant toward reconstructing other bacterial toxin and viral interactions under late endosome conditions. ? These easy yet powerful approaches of controlled immobilized construction and release are most certainly applicable toward other problem protein constructions where pH dependent changes occur that result in membrane insertion that dictate bacterial toxin and viral entry into the cell.